Yoshiko Banno

Sphingosine 1‐phosphate as a regulator of osteoclast differentiation and osteoclast–osteoblast coupling

Sphingosine 1‐phosphate (S1P), produced by sphingosine kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK1 and S1P in osteoclastogenesis using bone marrow‐derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF‐κB ligand (RANKL) upregulated SPHK1 and increased S1P production and secretion. SPHK1 siRNA enhanced and SPHK1 overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc1 and c‐Fos levels. Extracellular S1P had no effect in these cultures. These data suggest that intracellular S1P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S1P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase‐2 and PGE2 regulation. S1P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S1P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S1P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMS Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype. METHODS AND RESULTS SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice. CONCLUSION These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Sphingosine Kinase Isoforms Regulate Oxaliplatin Sensitivity of Human Colon Cancer Cells through Ceramide Accumulation and Akt Activation

The relationship between sphingosine kinase (SPHK), cellular ceramide concentration and chemosensitivity was investigated in human colon cancer cell lines. Among nine colon cancer cell lines, SPHK1 and SPHK2 activity and protein expression was highest in RKO cells and lowest in HCT116 cells. A viability assay revealed that HCT116 cells were sensitive to the effects of oxaliplatin (l-OHP), whereas RKO cells were resistant to those of l-OHP. Treatment with 5μg/ml l-OHP induced a marked time-dependent increase in various ceramides (C16, C24, C24:1) in HCT116 cells but not in RKO cells, as indicated by liquid chromatography/mass spectrometry. The increase in ceramide and caspase activation induced by l-OHP in the sensitive HCT116 cells was abolished by pretreatment with a neutral sphingomyelinase inhibitor, suggesting that the ceramide formation was due to the activation of neutral, rather than acid, sphingomyelinase. In contrast, in l-OHP-resistant RKO cells, treatment with an SPHK inhibitor or SPHK1 and SPHK2 silencing by RNA interference suppressed cell viability and increased caspase activity and cellular ceramide formation after l-OHP treatment. The elevated ceramide formation induced by SPHK inhibition and l-OHP was inhibited by fumonisin B1 but not myriocin, suggesting that ceramide formation was through the salvage pathway. Endogenous phosphorylated Akt levels were much higher in the resistant RKO cells than in the sensitive HCT116 cells. Either SPHK1 or SPHK2 silencing in RKO cells decreased phosphorylated Akt levels and increased p53 and p21 protein levels as well as poly(ADP-ribose) polymerase cleavage in response to l-OHP treatment. These findings indicate that SPHK isoforms and neutral sphingomyelinase contribute to the regulation of chemosensitivity by controlling ceramide formation and the downstream Akt pathway in human colon cancer cells.

Association of a polymorphism of the phospholipase D2 gene with the prevalence of colorectal cancer

Phospholipase D plays an important role in transmembrane signaling in a variety of cell types and its activity is increased in certain cancers, suggesting that it also contributes to tumorigenesis. A C→T transition at nucleotide 1814 of the human phospholipase D2 gene, which results in a Thr→Ile substitution at amino acid 577, was noted in the GenBank database. The relationship of this polymorphism to the prevalence of cancer of the esophagus, stomach, colon-rectum, lung, and breast in Japanese was investigated in a case-control study. The genotype of the phospholipase D2 gene was determined by the polymerase chain reaction with confronting two-pair primers. Multivariate logistic regression analysis with adjustment for age, gender, and smoking status revealed that the frequency of the T allele of the 1814C→T polymorphism was significantly higher in individuals with colorectal cancer than in controls. A significant association of the polymorphism with the prevalence of colorectal cancer was found in analyses assuming either dominant (TT+CT vs. CC) or additive (CT vs. CC) effects of the T allele, but the T allele was not associated with the prevalence of esophageal, gastric, lung, or breast cancer. The activities of phospholipase D in cell lysates or membrane fractions did not differ between cells transfected with cDNAs encoding the Thr-577 or Ile-577 variants of phospholipase D2. These results suggest that the phospholipase D2 gene is a susceptibility locus for colorectal cancer in Japanese individuals, although a functional effect of the 1814C→T (Thr577Ile) polymorphism was not detected.

Transcription factor specificity protein 1 (Sp1) is the main regulator of nerve growth factor‐induced sphingosine kinase 1 gene expression of the rat pheochromocytoma cell line, PC12

Sphingosine kinase (SPHK) is known to exert an anti‐apoptic role in various cells and cell lines. We previously reported that human brain is rich in SPHK1 ( Murate et al. 2001 ). After showing a high expression of SPHK1 in rat brain, we examined the gene expression mechanism using nerve growth factor (NGF) ‐stimulated rat PC12 cells. With RT–PCR, we found that both rat brain and PC12 utilized exon 1d mostly out of eight untranslated first exons. NGF induced an increase in SPHK enzyme activity and protein about double those in PC12 cells, and NGF‐induced SPHK1 mRNA was three times higher than in the control. The minimal 5′ promoter was determined, and TrkA specific inhibitor K252a inhibited the NGF‐induced promoter activity of SPHK1. The truncation or mutation of putative transcription factor‐binding motifs revealed that one specificity protein 1 (Sp1) binding motif of the 5′ region of exon 1d is prerequisite. Electrophoresis mobility shift assay confirmed the promoter analysis, indicating increased Sp1 protein binding to this motif after NGF treatment. Chromatin immunoprecipitation assay also showed the binding of Sp1 and the promoter region in vivo. These results suggest the signal transduction pathway from NGF receptor TrkA to transcription factor Sp1 protein binding to the promoter Sp1‐like motif in NGF‐induced rat SPHK1 gene expression.

Quantitative RT-PCR analysis of sphingolipid metabolic enzymes in acute leukemia and myelodysplastic syndromes

Quantitative RT-PCR analysis of sphingolipid metabolic enzymes in acute leukemia and myelodysplastic syndromes

Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10

Gelsolin expression is frequently downregulated in lung cancer and several types of different human cancers. To examine the effects of gelsolin restoration on tumorigenicity, we here stably expressed various levels of gelsolin via gene transfer in lung cancer cells (squamous cell carcinoma line, PC10). We observed the alterations in tumorigenicity in vivo when implanted in nude mice, and the changes in growth properties in vitro. As compared to parental cells and control clones, gelsolin transfectants highly reduced tumorigenicity and repressed cell proliferation. Moreover, we investigated bradykinin-induced responses in gelsolin-overexpressing clones, because agonist-stimulated activation of the phospholipases C (PLC)/protein kinase C (PKC) signal transduction pathway is critical for cell growth and tumorigenicity. Bradykinin promotes phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC and translocation of various PKC isoforms from the cytosolic fraction to the particulate fraction. Bradykinin treatment did not increase inositoltriphosphate (IP3) production and induce the membrane fractions of PKCα and PKCγ in gelsolin tranfectants, while it induced PIP2 hydrolysis and increased the fractions in parental and control clones. These results suggest that gelsolin suppressed the activation of PKCs involved in phospholipid signalling pathways, inhibiting cell proliferation and tumorigenicity.

Expression of the NF-κB Target Gene X-Ray-Inducible Immediate Early Response Factor-1 Short Enhances TNF-α-Induced Hepatocyte Apoptosis by Inhibiting Akt Activation

Using a cDNA microarray analysis, we identified x-ray-inducible immediate early response factor-1 (IEX-1) as a proapoptotic gene which was induced by TNF-α and also depend on NF-κB activation in Hc human hepatocytes. In these cells only the original form of IEX-1, termed IEX-1S, but not its longer transcript IEX-1L, was expressed. Overexpression of IEX-1S resulted in promotion of TNF-α-induced apoptosis in Hc cells expressing a mutant form of IκB. This proapoptotic action can be explained by its inhibitory findings on survival signals; inhibition of TNF-α-induced activation and expression of phosphatidylinositol 3-kinase (PI3K)/Akt, and also blockage of expression of Mcl-1, an antiapoptotic Bcl-2 family member which is located downstream of Akt, was inhibited by IEX-1S. LY 294002, an inhibitor of PI3K, increased IEX-1S expression induced by TNF-α and accelerated TNF-α-induced apoptosis in IκB-treated Hc cells. Overexpression of the dominant-negative Akt enhanced, but the constitutively active Akt suppressed, TNF-α-induced IEX-1S expression, suggesting that PI3K/Akt negatively regulated IEX-1S expression. These results demonstrate that NF-κB-dependent recruitment of IEX-1S may play a proapoptotic role in TNF-α-stimulated hepatocytes through blockage of the PI3K/Akt pathway. Moreover, the reciprocal cross-talk between IEX-1S and PI3K/Akt may closely be involved in the regulation of TNF-α-induced hepatocyte apoptosis.

Interferon-γ Decreases Ceramides with Long-Chain Fatty Acids: Possible Involvement in Atopic Dermatitis and Psoriasis

Ceramide (CER) with long-chain fatty acids (FAs) in the human stratum corneum (SC) is important for the skin barrier functions. Changes in the CER profile have been associated with abnormal permeability of dermatoses such as atopic dermatitis (AD) and psoriasis. In addition, interferon-γ (IFN-γ) has been known to be abundant in both AD and psoriatic skin lesions. In this study, we aimed to identify the mechanism underlying the alteration of FA chain length of CERs in these diseases. Mass spectrometry analysis of CERs in the SC showed that the proportion of CERs with long-chain FAs was significantly lower in AD and psoriasis patients than in healthy controls, and this reduction was more pronounced in psoriasis than in AD. Using cultured human keratinocytes and epidermal sheets, we found that only IFN-γ among various cytokines decreased the mRNA expression of elongase of long-chain fatty acids (ELOVL) and ceramide synthase (CerS), enzymes involved in FA chain elongation. Furthermore, quantitative analysis showed that IFN-γ decreased the levels of CERs with long-chain FAs. These results suggest that IFN-γ decreases CERs with long-chain FAs through the downregulation of ELOVL and CerS and that this mechanism may be involved in the CER profile alteration observed in psoriasis and AD.

Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells

Melanogenesis is a principal parameter of differentiation in melanocytes and melanoma cells. Our recent study has demonstrated that phospholipase D1 (PLD1) regulates the melanogenic signaling through modulating the expression of tyrosinase, the rate‐limiting step enzyme in the melanin biosynthesis. The current study was designed to gain more insight into the involvement of PLD1 in the regulation of melanogenesis. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on melanogenesis in B16 melanoma cells. It was shown that the melanin synthesis was induced in PLD1‐knockdowned cells, and also that the level of melanin synthesis was well correlated with increases in expression level of tyrosinase and its related proteins (Tyrp1 and Dct). Furthermore, the reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment malanoma cells with rapamycin, a potent inhibitor of mTOR effectively induced melanogenesis. The results obtained here provide possible evidence that PLD1 exerts a negative regulatory role in the melanogenic process through mTOR/S6K1 signaling.